Fig. 2
Changes in the properties of ALI multilayered co-cultures overtime. a and b Representative LSCM images of the a F-actin organization (in red) and b Ki67 protein expression (in green) in ALI multilayered co-cultures over 14-day growth. Full datasets for all time-points are reported in the Additional file 1. Scale bars: 20 μm (objective lens: 63×). a Cell nuclei were also stained with Hoechst 33342 (in blue). Z-stack images, here presented in orthogonal view, clearly demonstrate the multilayered structure of the in vitro models developed. b Z-stack images of the apical side of the cultures were reconstructed and are shown as three-dimensional projections. c Time-dependent changes in: ATP levels, percentage (%) of live A549 cells, TEER, % of LY passage and Papp values in ALI multilayered co-cultures grown up to 14 d. Data are shown as average ± standard error of the mean (nreplicates = 2; ntests = 3). The symbols (**) and (***) indicate statistically significant changes as compared to the values measured at 24 h (p < 0.01 and 0.001, respectively) (two-way ANOVA and Bonferroni post-test). d Western blot analysis of E-cadherin (epithelial marker), vimentin and fibronectin (mesenchymal markers) in A549 cells forming ALI multilayered co-cultures and cultured up to 14 d. The time-points examined were: 24 h, 48 h, 72 h, 7 d and 14 d. Abbreviations “n1” and “n2” indicate different experimental replicates. β-actin expression is also reported as protein loading control